Cytokines regulate cellular growth and differentiation, but are also important in regulating immune and inflammatory responses, and are critical in the pathogenesis of autoimmune diseases such as rheumatoid arthritis, systemic lupus erythematosus, inflammatory bowel disease, psoriasis, allergy and asthma. Targeting cytokines and cytokine signaling has led to successful new strategies in treating these diseases, underscoring the need to better understand the molecular basis of cytokine action as it relates to the pathogenesis of immune-mediated disease. A critical means through which cytokines exert their effect is activation of receptor-associated Janus kinases, or JAKs, and the activation of a family of transcription factors called STATs (signal transducers and activators of transcription); this has been the focus of our work for the last two decades. One important action of cytokines in which STAT proteins play a key role is the differentiation of different subsets of lymphocyte to attain distinct fates. CD4+ T follicular helper cells (TFH) are critical for the formation and function of B cell responses to infection or immunization, but also play an important role in autoimmunity. Because type I IFNs are often generated in immune responses, we set out to investigate whether these factors are relevant to TFH cell differentiation. We found that type I IFNs induced Bcl6 expression, the master regulator transcription factor for TFH cells, and CXCR5 and programmed cell death-1 (encoded by Pdcd1), key surface molecules expressed by TFH cells. In contrast, type I IFNs failed to induce IL-21, the signature cytokine for TFH cells. The induction of Bcl6 was regulated directly by Stat1, which bound to the Bcl6, Cxcr5, and Pdcd1 loci. Previously, we demonstrated the importance of Stat3 for Th17 differentiation and, in collaboration with NIH colleagues, showed that this was relevant to the pathogenesis of Hyperimmunoglobulin E or Jobs syndrome. Using patient-derived mutations, we generated a mouse model of Jobs syndrome and documented the role of dominant negative Stat3 mutations in dysregulation of Th17 cells but also barrier function in the gut. We also showed that treatment of this disorder in the mouse model with stem cell transplantation partially corrected these deficits. In other collaborations, we identified novel approaches to targeting Th17 cells for the treatment of autoimmune disease. In collaboration, we also identified the kinase DYRK1A as an important regulator of Th17 versus Treg differentiation. Interleukin-6 (IL-6) and IL-27 both employ Stat1 and Stat3 for signaling. We assessed the relative contributions of STAT1 and STAT3 using genetic models and chromatin immunoprecipitation-sequencing (ChIP-seq) approaches. We found an extensive overlap of the transcriptomes induced by IL-6 and IL-27 and few examples in which the cytokines acted in opposition. Using STAT-deficient cells and T cells from patients with gain-of-function STAT1 mutations, we demonstrated that STAT3 is responsible for the overall transcriptional output driven by both cytokines, whereas STAT1 is the principal driver of specificity. STAT1 cannot compensate in the absence of STAT3 and, in fact, much of STAT1 binding to chromatin is STAT3-dependent. IL-9 is an important cytokine that also has important roles in intestinal barrier function. A subset of CD4 T cells that preferentially produce IL-9 are termed Th9 cells. In collaboration with the Siegel lab, we showed that TL1A potently promotes generation of Th9 cells through an IL-2 and STAT5-dependent mechanism, unlike the TNF-family member OX40, which promotes Th9 through IL-4 and STAT6. We had previously shown that furin was an important Stat target gene that was critical to immune tolerance by processing TGFb. In more recent work, we showed that T cell-expressed furin is essential for normal Th1 function. In related work, we investigated the role of EZH2, a component of the Polycomb complex in helper T cells. FOXP3 is critical factor for preserving immune tolerance and regulatory T cell function. FOXP3 associates with EZH2 to mediate gene repression and suppressive function. We found that deletion of Ezh2 in CD4 T cells resulted in reduced numbers of Treg cells. We found that both Ezh2-deficient Treg cells and T effector cells were functionally impaired in vivo: Tregs failed to constrain autoimmune colitis, and T effector cells neither provided a protective response to T. gondii infection nor mediated autoimmune colitis. We also studied the function of the BET family proteins, BRD4, and found that BRD4 occupies widespread genomic regions in mouse cells and directly stimulates elongation of both protein-coding transcripts and noncoding enhancer RNAs. BRD4 interacts with elongating Pol II complexes and assists Pol II in progression through hyperacetylated nucleosomes by interacting with acetylated histones via bromodomains. Thus, BRD4 is involved in multiple steps of the transcription hierarchy, primarily by facilitating transcript elongation both at enhancers and on gene bodies independently of P-TEFb. IL-10 is another key mechanism that preserves immune tolerance. We found that innate immune cells, specifically NK cells, switch from predominantly producing IFN-g, a cytokine with proinflammatory and antimicrobial functions, to producing IL-10 during the course of viral infection. In contrast to the sustained open profile of the Ifng gene, during the course of infection NK cells acquired permissive histone modifications in the IL10 locus. This occurred concomitant with NK cell proliferation. Ongoing studies are investigating how the chromatin landscape is modified in innate lymphoid cells versus T cells and which factors drive these differences. A major focus of the laboratorys efforts over the past several years has been to approach the issue of helper T cell specification using new tools that allow genomic views of differentiating cells. A powerful technique has been chromatin immunoprecipitation and massive parallel sequencing (ChIP-seq). We used this technology to map active enhancer elements in T helper 1 (Th1) and Th2 cells. Our data establish that STAT proteins have a major impact on the activation of lineage-specific enhancers and the suppression of enhancers associated with alternative cell fates. More recently, we have used this approach to identify regions of the genome that are subject to intense regulation, so-called stretch or super-enhancers. We analyzed maps of mouse T-cell SEs as a non-biased means of identifying key regulatory nodes involved in cell specification and found that cytokines and cytokine receptors were the dominant class of genes exhibiting SE architecture in T cells. The locus encoding Bach2, a key negative regulator of effector differentiation, emerged as the most prominent T-cell SE, revealing a network in which SE-associated genes critical for T-cell biology are repressed by BACH2. Disease-associated single-nucleotide polymorphisms for immune-mediated disorders, including rheumatoid arthritis, were highly enriched for T-cell SEs versus typical enhancers or SEs in other cell lineages. Treatment of T cells with the Janus kinase inhibitor tofacitinib disproportionately altered the expression of rheumatoid arthritis risk genes with SE structures. This year we also reported the development of a new tool for Chip-seq analysis: PAPST, or Peak Assignment and Profile Search Tool. This new resource integrates both gene-centric and peak-centric co-localization analysis into a single package.